The present invention generally relates to the use of recombinant DNA technology to create a microorganism capable of producing antigens reactive with antibodies developed in response to Treponema pallidum. The production, by genetically engineered microorganisms, of the immunodominant 47-48 kilodalton surface antigen of Treponema pallidum, generally referred to as the 47 kilodalton surface antigen, is a particular object of the present invention.
Infection by Treponema pallidum is the well-established cause of syphillis. Syphillis, despite the continued availability of effective antibiotics, continues to result in worldwide misery and morbidity. The inability to establish in vitro cultures of Treponema pallidum has restricted research studies of this organism. This microorganism is usually propagated by in vivo culture in hamster lymph nodes or rabbit testes, which are useful but not methods conducive to large-scale production of Treponema pallidum or the antigens thereof, particularly pure antigens. It has proven difficult to obtain Treponema pallidum antigens free of hamster or rabbit antigens from Treponema pallidum cultured in vivo. Thus, purified treponemal antigens have not been available from traditional techniques of microorganism production.
More modern techniques of molecular biology have been applied in relation to characterization and detection of Treponema pallidum, Treponema pallidum antigens and infection by Treponema pallidum. Various polyclonal and monoclonal antibodies to Treponema pallidum antigens have also been prepared.
U.S. Pat. No. 4,514,498, issued to Kettman and Norgard, Apr. 30, 1985 described numerous monoclonal antibodies directed toward antigens of T. pallidum. Two hybridoma clones 8G2 (ATCC HB8134) and 11E3 produced antibodies specific for the 47 kDa surface antigen of T. pallidum. These particular two antibodies were used in the present invention for the selection of E. coli clones producing the 47 kDa antigen.
Norgard et al. (1983) (Infect. and Immun., V 42, pp 435-445) described construction of hybrid pBR322 plasmid clone banks in E. coli K-12 representing the entire--Treponema pallidum genome. Rabbit anti-T. pallidum serum was used in a radioimmuno-colony blot assay to detect E. coli clones synthesizing T. pallidum antigens. One clone was found to encode a gene product of 44 kDa. Either immune rabbit serum or human syphilitic serum was used to detect the E. coli clones producing the T. pallidum antigen. The methodological difference between this reference and the present disclosure begins to distinguish the present invention. Additionally, it has been found that monoclonal antibodies specific for the T. pallidum 47 kDa surface antigen were unable to react with the 44kDa antigen.
Stamm et al., (1982) (Infect. and Immun., V 36, pp 1238-1241) described the establishment of a T. pallidum DNA clone bank. T. pallidum DNA was cleaved with Bam HI endonucleus and the cleavage fragments ligated to Bam HI-cleaved plasmid pBR322. Four transformed E. coli clones were noted as generally producing T. pallidum antigens.
Stamm et al. (1983) (Infect. and Immun., V 41, pp 709-721) described the expression of Treponema pallidum Antigens in Escherichia coli K-12. Stamm et al. (1983) describe use of a pBR322 bank of recombinant plasmids harboring Treponema pallidum DNA inserts in Escherichia coli and identification of clones expressing Treponema pallidum antigens. The molecular weights of antigens produced by plasmid pLV32-transformed E. coli. were 25 kDa, 35 kDa and 39 kDa in size. An E. coli clone bearing plasmid pLV55 encoded a 35 kDa, T. pallidum protein antigen. An object of the Stamm et al. (1983) reference was to produce T. pallidum surface antigens. In the Stamm et al. (1983) reference whole human or rabbit serum against syphillis was used to select transformed E. coli clones.
Van de Donk et al. (1984) (in Innovations in Biotechnology, ed. by Houwink, et al., Elsevier Science, Amsterdam) described the development of hybridoma cell lines producing antibodies directed against T. pallidum antigens. It also discloses that certain monoclonal antibodies obtained from the hybridoma cell lines reacted with a 46 kDa and a 44 kDa polypeptide antigen of Treponema pallidum. Furthermore, the reference indicated that these monoclonal antibodies may be used for the purification of Treponema pallidum antigens produced by E. coli carrying Treponema pallidum recombinant DNA, as previously suggested by Norgard et al. (1983) (Infect. and Immun., V 42, pp 435-445).
Van Embden et al., (1983) (Infect and Immun., V 42, pp 187-196) described the production of E. coli K-12 clones transformed with plasmids bearing T. pallidum DNA. E. coli transformants were noted producing a variety of T. pallidum polypeptide antigens, including polypeptides with apparent sizes of 35 kDa, 41 kDa, 44 kDa, and 58 kDa, among others.
Coates et al. (1985) (Abstracts of the 85th Annual Meeting of the Amer. Soc. for Microbiol., Abstract C29, p 304) described an E. coli transformant producing an 18 kDa T. pallidum polypeptide antigen.
Fehniger et al., (1985) (Abstracts of the 85th Annual Meeting of Amer. Soc. for Microbiol., Abstract B156, p 44) described a 38 kDa hydrophobic T. pallidum antigen as produced by an E. coli transformed with a recombinant plasmid.
Rodgers et al., (1985) (Abstracts of the 85th Annual Meeting of Amer. Soc. for Microbiol., Abstract C30, p 305) described evaluation of the reactivity of a 37 kDa polypeptide T. pallidum antigen produced by an E. coli transformant.
PCT patent application (1984) number PCT/US83/01718 (International Publication Number WO84/01961 entitled "Recombinant DNA Derived Antigens of Treponema Pallidum" by Lovett) described E. coli transformant clones containing plasmid-bound T. pallidum DNA and expressing T. pallidum peptide antigens. Various clones produced antigens with molecular weights or weight ranges of: 16-20 kDa; 43 kDa; 37-46 kDa; 18-23 kDa; 150 kDa; or 180 kDa.
Potentially important antigens or immunogens of T. pallidum have been identified in several laboratories (Alderete et al. (1980) Infect. Immun., V 30, pp 814-823; Baker-Zander et al. (1985) (J. Infect. Dis., V 151, pp 264-272; Baker-Zander et al., (1983) Infect. Immun., V 42, pp 634-638; Baker-Zander et al. (1984) Infect. Immun., V 46, pp 116-121; Hanff et al., (1982) J. Immunol., V 129, pp 1287-1291; Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420; Lukehart et al., (1982) J. Immunol., V 129, pp 833-838; Lukehart et al. (1986) Sex. Trans. Dis., V 13, pp 9-15; Lukehart et al. (1985) J. Immunol., V 134, pp 585-592; Marchitto et al. (1984) Infect. Immun., V 45, pp 660-666,; Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176; Moskophidis et al. (1984) Infect. Immun., V 43, pp 127-132; Norris et al. (1984) J. Immunol., V 133, pp 2686-1692; Penn et al. (1985) J. Gen. Microbiol., V 131, pp 2349-2357; Strugnell et al. (1986) Infect. Immun., V 51, pp 957-960; Thornburg et al. (1983) Infect. Immun., V 42, pp 623-627; Thornburg et al. (1985) Genitourin, Med., V 61, pp 1-6; and van Eijk et al. (1982) J. Microbiol., V 48, pp 486-497). Potential biological significance was previously assigned to a major, immunogenic surface antigen of T. pallidum having a molecular mass of 47 kilodaltons (kDa) (Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420; and Marchitto et al. (1984) Infect. Immun., V 45, pp 660-666). Biologically active monoclonal antibodies (mAbs), in combination with various in vitro and in vivo assays, were used to characterize the immunogen (Jones et al. (1984) J. Exp. Med., V 160, pp 1404-1420; Marchitto et al. (1984) Infect. Immun., V 45, pp 660-666; Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176; and Norgard et al. (1984) J. Clin. Microbiol., V 20, pp 711-717). The 47 kDa antigen was shown to be: (i) surface-associated; (ii) abundant (Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420; Marchitto et al. (1984) Infect. Immun., V 45, pp 660-666; and Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176); (iii) highly immunogenic in both rabbits and humans (Baker-Zander et al. (1985) J. Infect. Dis., V 151, pp 264-272; Hanff et al. (1982) J. Immunol., V 129, pp 1287-1291; Jones et al. (1984) J. Exp. Med., V 160, pp 1404-1420; Lukehart et al. (1986) Sex. Trans. Dis., V 13, pp 9-15; Strugnell et al. (1986) Infect. Immun., V 51, pp 957-960; and van Eijk et al. (1982) J. Microbiol., V 48, pp 486-497); (iv) proteinaceous; (v) found in at least three subspecies of pathogenic T. pallidum, the etiological agents of veneral syphilis, endemic syphilis, and yaws; and (vi) absent in nonpathogenic, saprophytic treponemes (Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420; Marchitto et al. (1984) Infect. Immun., V 45, pp 660-666; Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176; and Norgard et al. (1984) J. Clin. Microbiol., V 20, pp 711-717). Anti-47 kDa mAbs possess diagnostic value; they bind strongly in immunofluorescence assays to T. pallidum isolated from human syphilitic lesions. Anti-47 kDa mAbs also partially block the attachment of T. pallidum to host cells in vitro (Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420). Anti-47 kDa mAbs bind to T. pallidum in the T. pallidum immobilization (TPI) assay, resulting in complement-dependent immobilization of motile organisms and these mAbs in the present of complement neutralize (kill) T. pallidum in the in vitro-in vivo neutralization test of Bishop and Miller (Bishop et al. (1986) J. Immunol., V 117, pp 197-207; Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420; and Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176).
The potential biological significance of the 47 kDa immunogen is supported by work on similar or identical immunogens. Lukehart et al. (Lukehart et al. (1982) J. Immunol., V 129, pp 833-838) and Baker-Zander and Lukehart (Baker-Zander et al. (1983) Infect. Immun., V 42, pp 634-638; and Baker-Zander et al. (1984) Infect. Immun., V 46, pp 116-121) reported that a 48 kDa immunogen of T. pallidum contained T. pallidum-specific epitopes that could be detected in the treponemal pathogens T. pallidum, T. pertenue (yaws organism), T. paraluis-cuniculi (agent of rabbit veneral spirochaetosis) and T. hyodysenteriae (agent of swine dysentery). Lukehart also observed an early and significant humoral response to the 47 kDa immunogen of T. pallidum in patients infected with T. carateum (pinta organism) (S. A. Lukehart, personal communication). Hanff et al. (Hanff et al., (1982) J. Immunol., V 129, pp 1287-1291) showed an early humoral immune response during human syphilis to T. pallidum antigens having molecular masses from 45-47 kDa. Baker-Zander et al. (Baker-Zander et al. (1985) (J. Infect. Dis., V 151, pp 264-272) confirmed the strong reactivity of syphilitic sera early in the course of human infection to a 48 kDa antigen of T. pallidum. This early and significant humoral immune response also was observed in experimental rabbits (Lukehart et al. (1986) Sex. Trans. Dis., V 13, pp 9-15). van Eijk and van Embden (van Eijk et al. (1982) J. Microbiol., V 48, pp 486-497) reported an early humoral immune response to a 46 kDa T. pallidum immunogen among humans with primary syphilis and later stages of the disease. Strugnell et al. (Strugnell et al. (1986) Infect. Immun., V 51, pp 957-960) reported that the most vigorous humoral immune response detectable early in the experimental rabbit was directed against a polypeptide of 47 kDa. Thornburg and Baseman (Thornburg et al. (1983) Infect. Immun., V 42, pp 623-627); and Thornburg et al. (Thornburg et al. (1985) Genitourin. Med., V 61, pp 1-6) also described a major 45 kDa immunogen of T. pallidum, which was found in T. pallidum and T. pertenue. Penn et al. (Penn et al. (1985) J. Gen. Microbiol., V 131, pp 2349-2357) recently concluded that T. pallidum contains an immunodominant, 47 kDa major outer membrane protein. Additionally, our earlier findings (Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176; and Norgard et al. (1984) J. Clin. Microbiol., V 20, pp 711-717) that anti-47 kDa mAbs can be used diagnostically to detect relatively few T. pallidum has been confirmed by Lukehart et al. (Lukehart et al. (1985 ) J. Immunol., V 134, pp 585-592) and Hook et al. (Hook et al. (1985) J. Clin. Microbiol., V 22, pp 241-244). Hook et al. (Lukehart et al. (1986) Sex. Trans. Dis., V 13, pp 9-15) also reported a correlation between early immune clearance of infecting T. pallidum and healing of the primary lesion in the experimental rabbit; it was postulated that primary lesion healing may be influenced by antibody directed against immunodominant molecules, such as the 47 kDa immunogen (Lukehart et al. (1986) Sex. Trans. Dis., V 13, pp 9-15).
Using serum from T. pallidum-infected rabbits or murine mAbs directed against the 47 kDa immunogen of T. pallidum, it was previously reported that the 47 kDa immunogen was pathogen-specific (Jones et al., (1984) J. Exp. Med., V 160, pp 1404-1420; Marchitto et al. (1984) Infect. Immun., V 45, pp 660-666; Marchitto et al. (1986) Infect. Immun., V 51, pp 168-176; and Norgard et al. (1984) J. Clin. Microbiol., V 20, pp 711-717); the presence of an analogous 47 kDa antigen was not detected in the nonpathogenic trephonemes T. phagedenis biotype Reiter, T. denticola, T. scoliodontum, T. vincentii, or T. refringens using immune rabbit serum or mAbs directed against the 47 kDa immunogen. Lukehart et al. (Lukehart et al., (1982) J. Immunol., V 129, pp 833-383; Lukehart et al. (1985) J. Immunol., V 134, pp 585-592) postulated that a 48 kDa T. pallidum immunogen may possess "pathogen-specific" determinants may be located on separate polypeptides which co-migrate in polyacrylamide gels. When either a 3.85 kb HindIII DNA fragment of the 5.4 kb encoding sequence or intact hybrid plasmid DNA (containing the entire 5.4 kb encoding sequence) were used as DNA hybridization probes under moderate or low DNA hybridization stringency, no hybridization with any homologous DNA fragment of the nonpathogenic treponemes was observed. Further, mAb C2-1 of Lukehart, directed against a "common" treponemal epitope of a 47 kDa immunogen (Hook et al. (1985) J. Clin. Microbiol., V 22, pp 241-244; and Lukehart et al. (1985) J. Immunol., V 134, pp 585-592), failed to react with the 47 kDa immunogen-expressing clones of the present invention, while the pathogen-specific mAb H9-1 (Hook et al. (1985) J. Clin. Microbiol., V 22, pp 241-244, Lukehart et al. (1985) J. Immunol., V 134, pp 585-592) reacted with these clones. Thus, genetic and immunologic data provided here support the existence of a pathogen-specific 47 kDa immunogen.
The cloning and expression of the T. pallidum 47 kDa immunogen gene in Escherichia coli is described herein as a component of the present invention.
The contents of the above-described references are incorporated by reference herein for their descriptions of microorganisms, materials and methods established in the field.